CHARACTERIZATION OF UREOLYTIC BACTERIA ISOLATED FROM LIMESTONE CAVES OF SARAWAK AND EVALUATION OF THEIR EFFICIENCY IN BIOCEMENTATION By ARMSTRONG IGHODALO OMOREGIE A thesis presented in fulfilment of the requirements for the degree of Master of Science (Research) Faculty of Engineering, Computing and Science SWINBURNE UNIVERSITY OF TECHNOLOGY 2016 ABSTRACT The aim of this study was to isolate, identify and characterise bacteria that are capable of producing urease enzyme, from limestone cave samples of Sarawak. Little is known about the diversity of bacteria inhabiting Sarawak’s limestone caves with the ability of hydrolyzing urea substrate through urease for microbially induced calcite precipitation (MICP) applications. Several studies have reported that the majority of ureolytic bacterial species involved in calcite precipitation are pathogenic. However, only a few non-pathogenic urease-producing bacteria have high urease activities, essential in MICP treatment for improvement of soil’s shear strength and stiffness. Enrichment culture technique was used in this study to target highly active urease- producing bacteria from limestone cave samples of Sarawak collected from Fairy and Wind Caves Nature Reserves. These isolates were subsequently subjected to an increased urea concentration for survival ability in conditions containing high urea substrates. Urea agar base media was used to screen for positive urease producers among the bacterial isolates. All the ureolytic bacteria were identified with the use of phenotypic and molecular characterizations. For determination of their respective urease activities, conductivity method was used and the highly active ureolytic bacteria isolated comparable with control strain used in this study were selected and used for the next subsequent experiments in this study. Effects of cultural conditions on urease activity and evaluation of biocementation potential of these locally selected ureolytic isolates were also performed. Out of the ninety bacteria subcultured from enriched cultures containing the cave samples, thirty-one bacterial isolates were selected based on their respective abilities of producing urease enzyme by completely turning the colour of urea agar base medium from yellow to pink in comparison to other isolated urease producing bacteria and the control strain (Sporosarcina pasteurii, DSM33) used in this study. The microscopic analysis using Gram staining technique showed that majority of the bacterial isolates were Gram-positive bacteria while only three of the isolates were Gram-negative bacteria. In addition, majority of the bacterial cells were rod-shaped except for one bacterial isolate which was a coccus. Endospore staining test results indicate also indicated that all except one isolate were spore forming bacteria. i The BLAST results from molecular characterization of the ureolytic isolates suggested that they were closely related to bacteria from the Sporosarcina pasteurii group, Pseudogracilibacillus auburnensis group, Staphylococcus aureus group, Bacillus lentus group, Sporosarcina luteola group and Bacillus fortis group when compared to the 16S rRNA sequencing data in NCBI nucleotide BLAST database. Specific urease activity determination from the calculation of conductivity and urease activity showed that out of all the bacterial cultures, bacterial isolates designated as NB33, LPB21, NB28, NB30 and the control strain had 19.975, 23.968, 19.275, 20.091 and 17.751 mM urea hydrolysed.min-1.OD-1 respectively, suggesting they had the highest specific urease activities when compared to the rest isolates. The effect of cultural conditions on urease activities involving the aforementioned local isolates and control strain showed that incubated these conditions: at 25 to 30oC; pH 6.5 to 8.0; incubation period at 24 hr; and urea concentration of 6 to 8%, maximum specific urease activities for the selected ureolytic bacteria isolates and control strain were obtained. The biocement treatment test using isolates NB33, LPB21, NB28, NB30 and the control strain on poorly graded soil clearly showed that MICP is microbially induced and not chemically induced. The results presented in this study showed that out of all the sand columns treated, all except the columns containing negative control (only cementation solution) had calcium carbonate precipitation shown on the top surfaces of their respective columns. Each column treated with microbial cultures and cementation solution (containing 1 M or urea and CaCl2) were able to bind the sand particles together. However, it was observed that there was higher cementation level at positions close to the injection points which resulting in more calcite contents to be obtained at this layers of the biocemented sands. Based on the surface strength using penetrometer test and compressive strength using UCS test, samples treated with isolates LPB21 and NB28 showed significant strengths when compared to other isolates, consortia, and the control strain. However, the rest isolates showed similar performance with the control strain. The application of these newly isolates highly active ureolytic bacteria can be used to for other MICP treatments in civil and geotechnical industries. The findings in this study suggest that the isolated ureolytic bacteria (NB28, LPB21, NB33, and NB30) have the potential to be used as alternative microbial MICP agents for biocement applications. ii ACKNOWLEDGEMENT Foremost, I would like to express my deepest gratitude to my principal coordinating supervisor: Assoc. Prof Dr Peter Morin Nissom (Associate Dean, Science) for all the valuable discussion, brainstorm, helpful advice, critics, challenges and encouragements throughout this research study. His overwhelming supervision made me develop new insights and ideas during this research. His quest for “high-quality work”, made me stay active, focused and enthusiastic. He also provided critical reviews of my experiments and writing, prompting me to improve problem solving and writing skills. I would also like to thank my associate supervisor: Dr Irine Runnie Ginjom for her insightful discussion and comments on my experimental progress. Her invaluable advice, co- supervision, and encouragement throughout this study helped made this thesis a success. I would like to gratefully acknowledge Assoc. Prof Dr Dominic Ek Leong Ong (Director, Swinburne Sarawak Research Centre for Sustainable Technologies) and Dr Ngu Lock Hei (Course coordinator, Chemical Engineering Department) for their financial support (SSRG) used to partially fund my research project. I am thankful for the continuous moral support and helpful discussion from Assoc. Prof Dr Dominic Ek Leong, especially with the idea of going to the caves to screen for calcite-precipitating microorganisms. I extend my appreciation to Sarawak Biodiversity Centre (SBC) and Sarawak Forestry Department (SFD) for issuing the permits (SBC-RA-0102-DO and NCCD.907.4.4 [JLD.11]-37) which enabled me to collect samples from Fairy Cave (N 01°22’53.39” E 110°07’02.70”) and Wind Cave (N 01°24’54.20” E 110°08’06.94”) Nature Reserves, located in Bau, Kuching Division, Sarawak, Malaysia. The collection of the samples from these extreme environments to conduct biological research stipulated the potentials of screening, identifying and characterising highly active isolated ureolytic bacteria. I am thankful to Dr Paul Mathew Neilsen, Associate director of graduate studies and research education. His thoughtful guidance and warm encouragement, especially during my confirmation of candidature helped make me achieve my research goals. I am sincerely grateful for his continual willingness of finding time out of his busy schedule to meet me and discuss on how I could tackle research challenges and improve my research study. iii I would also like to acknowledge Assist. Prof Salwa Al-Thawadi, Dr Ralf Cord- Ruwisch, PD Dr David Schleheck and Assist. Prof Leon van Paassen for providing indispensable guidance on how to measure urease activity, the appropriate way of determining specific urease activity and selective investigation of cultural conditions on urease activities. I am very thankful for taking your time to reply my inquiries via emails and researchgate.net. I am thankful to the science laboratory officers and technicians: Chua JiaNi, Nurul Arina Salleh, Cinderella Sio and Marclana Jane Richard, for providing me with experimental materials and allowing me to make use of some apparatus during the course of my research study. Without their enormous assistance, my research would not have been completed on time. An exceptional gratitude goes to Hasina Mohammed Mkwata for being a helpful research lab mate and an amazing girlfriend. Her assistance while I carried out my experiment, specifically during the measurement of conductivity, biomass concentration and effect of cultural conditions on urease activity made my experiments very convenient. I also extend my appreciation to Ghazaleh Khoshdelnezamiha for playing a significant role during the in vitro biocement test. Her efforts and a keen interest in my research made my experiment successful. An extensive appreciation goes to Dr Noreha Mahidi and Holed Juboi for their vehement assistance during molecular characterization of the isolated ureolytic bacteria. It was a pleasure working with her. Big thanks also go to my fellow lab colleagues: Nurnajwani Senian and Ye Li Phua, for providing assistance during sample collection and when I conducted my experiments in the laboratory. I would like to thank